Identification of genetic markers associated with growth and morphology quality in senegalese sole (solea senegalensis) to boost aquaculture production

The Senegalese sole (Solea senegalensis) is one of the most valuable flatfish in aquaculture in Southern Europe and although the production has grown exponentially in the last decade, reproduction success, health status, and the improvement of growth rates and morphology quality still remain as important limitations for industrial exploitation. The development of breeding programs is a fundamental tool to solve these problems but requires estimate genetic components of economically valuable traits such as those growth and morphology related under industrial conditions. By other side, the advances in genomics provide new highly powerful analysis tools to determine more accurately genetic components productive traits. In this thesis, new genomic tools and molecular markers as well as the genetic components of growth and morphology quality traits have been developed in sole. For this purpose, firstly, a highdensity SNP genetic map and a de novo sole genome assembly were generated. Later, genetic and physical maps were anchored and integrated into 21 linkage groups (SseLGs) corresponding to the expected number of chromosomes of this species. Genetic map was bigger in female than male (1.49) observing also a different recombination rate landscape between sexes. The integrated physical map obtained was used for an association study to identify sex-linked markers. Seven families were analyzed using ddRAD and 30 significant sex-associated SNP markers located onto SseLG18 were identified. Searching for candidate genes for sex determination identified the follicle stimulating hormone receptor (fshr) that it was located within a hot recombination region although with an incomplete penetrance. In addition to SNP markers, genome information was used for searching and identifying SSR markers. Hence, 108 new SSR markers distributed throughout the genome were identified. They were structured in 13 PCR Multiplex assays (with up to 10‐plex) and the amplification conditions were optimized and validated with a high‐quality score. A subset of 40 highly polymorphic markers were selected to optimize four supermultiplex PCR Multiplex assays (8-11 SSRs per assay) were designed for use in pedigree analysis. Moreover, a new integrated genetic map with 229 SSRs distributed in 21 SseLGs was created by in silico genomic analysis. Both maps generated in this thesis were used to carry out evolutive genome studies in flatfish to identify lineage-specific Robertsonian fusions and several other rearrangements that explain changes in chromosome number in the karyotype of Pleuronectiformes.To investigate the genetic components of growth and morphology-related traits, estimates for different variables were determine before on-growing (400 days) and at harvest (800 days). Growth-related traits such as body weight (W), standard length (SL), width (W) and body area (A) showed high heritabilities (ranging from 0.568 to 0.609 at 400 d and from 0.424 to 0.500 at 800 d) with very high genetic correlations (>0.94) at both ages. With respect to morphology quality traits, six quality predictors including ellipticity (E), body height at the pectoral fin base (BHP), body maximum height (BMH) and caudal peduncle height (CPH) and two ratios (BMH/BHP and BMH/CPH) were evaluated. Results showed high heritabilities (0.463-0.774) for E, BHP, BMH and CPH which were higher at 400 d than 800 d. In contrast, the BMH/BHP and BMH/CPH ratios showed low-moderate heritabilities (0.144-0.306). High positive correlations (>0.95) were found between growth traits and the three heights, which decreased with age. In contrast, ellipticity showed negative and medium-high genetic correlations with growth traits and heights, indicating that fish selected for larger size will also be less elliptical. Finally, an association study to find genetic markers linked to growth traits was carried out. A low-density DNA chip was designed and validated for 49 SNPs distributed in 17 SseLGs. The analysis of fast and slow-growing families identified two significant markers within the general transcription factor 3C polypeptide 4 and the mitochondrial fission process protein 1. All these results provide powerful tools for genomic analysis as well as genetic highly valuable information to design genetic breeding programs in Senegalese sole to optimize to boost the industrial production in aquaculture

Chromosome anchoring in Senegalese sole (Solea senegalensis) reveals sex-associated markers and genome rearrangements in flatfish

Abstract The integration of physical and high-density genetic maps is a very useful approach to achieve chromosome-level genome assemblies. Here, the genome of a male Senegalese sole (Solea senegalensis) was de novo assembled and the contigs were anchored to a high-quality genetic map for chromosome-level scaffolding. Hybrid assembled genome was 609.3 Mb long and contained 3403 contigs with a N50 of 513 kb. The linkage map was constructed using 16,287 informative SNPs derived from ddRAD sequencing in 327 sole individuals from five families. Markers were assigned to 21 linkage groups with an average number of 21.9 markers per megabase. The anchoring of the physical to the genetic map positioned 1563 contigs into 21 pseudo-chromosomes covering 548.6 Mb. Comparison of genetic and physical distances indicated that the average genome-wide recombination rate was 0.23 cM/Mb and the female-to-male ratio 1.49 (female map length: 2,698.4 cM, male: 2,036.6 cM). Genomic recombination landscapes were different between sexes with crossovers mainly concentrated toward the telomeres in males while they were more uniformly distributed in females. A GWAS analysis using seven families identified 30 significant sex-associated SNP markers located in linkage group 18. The follicle-stimulating hormone receptor appeared as the most promising locus associated with sex within a region with very low recombination rates. An incomplete penetrance of sex markers with males as the heterogametic sex was determined. An interspecific comparison with other Pleuronectiformes genomes identified a high sequence similarity between homologous chromosomes, and several chromosomal rearrangements including a lineage-specific Robertsonian fusion in S. senegalensis

Development of whole-genome multiplex assays and construction of an integrated genetic map using SSR markers in Senegalese sole

The Senegalese sole (Solea senegalensis) is an economically important flatfish species. In this study, a genome draft was analyzed to identify microsatellite (SSR) markers for whole-genome genotyping. A subset of 224 contigs containing SSRs were preselected and validated by using a de novo female hybrid assembly. Overall, the SSR density in the genome was 886.7 markers per megabase of genomic sequences and the dinucleotide motif was the most abundant (52.4%). In silico comparison identified a set of 108 SSRs (with di-, tetra- or pentanucleotide motifs) widely distributed in the genome and suitable for primer design. A total of 106 markers were structured in thirteen multiplex PCR assays (with up to 10-plex) and the amplification conditions were optimized with a high-quality score. Main genetic diversity statistics and genotyping reliability were assessed. A subset of 40 high polymorphic markers were selected to optimize four supermultiplex PCRs (with up to 11-plex) for pedigree analysis. Theoretical exclusion probabilities and real parentage allocation tests using parent-offspring information confirmed their robustness and effectiveness for parental assignment. These new SSR markers were combined with previously published SSRs (in total 229 makers) to construct a new and improved integrated genetic map containing 21 linkage groups that matched with the expected number of chromosomes. Synteny analysis with respect to C. semilaevis provided new clues on chromosome evolution in flatfish and the formation of metacentric and submetacentric chromosomes in Senegalese sole.This study was funded by project RTA2017-00054-C03-01, RTA2017-00054-C03-02 and RTA2017-00054-C03-03 funded from MCIU/AEI/FEDER, UE. IGC is funded by a predoctoral fellowship from INIA.Ye